Many anticancer drugs act by inducing apoptosis (20). The rapid progress in understanding mechanisms underlying apoptosis may present opportunities to harness the cellular death machinery for the benefit of treating human diseases such as cancer (20, 35). Ideally, therapeutic strategies targeting an apoptotic pathway(s) should selectively kill cancer but not other cells. At present, however, this remains a very challenging objective.
Breast cancer is the second leading cause of cancer-related deaths in women (23). Each year more than 180,000 women in the United States are diagnosed with breast cancer. Currently, effective drug treatment for breast cancer is somewhat limited. Since many early stage breast tumors express the estrogen receptor (ER), and depend on estrogen for their optimal growth, anti-estrogens (ER antagonists) have been widely used in the treatment of ER+ tumors (5, 41). Anti-estrogens, however, are not effective in ER− tumors. Also, tumors that are initially ER+ may lose ER expression and become independent of estrogen for their growth and refractory to anti-estrogen therapy.
Currently, the primary treatment of localized breast cancer is either breast-conserving surgery and radiation or mastectomy with or without breast reconstruction. Systemic adjuvant therapies are also employed to eradiate microscopic deposits of cancer cells that may have spread or metastasized from the primary tumor. Systemic adjuvant therapies include chemotherapy and hormonal therapy. Radiation is also used as a local adjuvant treatment to eradicate cancer cells in the chest wall or regional lymph nodes after mastectomy (reviewed in 54). Major acute and long-term side effects of adjuvant treatments include premature menopause, weight gain, mild memory loss and fatigue.
Apoptosis or programmed cell death is a fundamental cellular process where the affected cell dies by actively executing a coordinately regulated death program (11, 18). For multicellular organisms (e.g. mammals) apoptosis plays important roles in normal development, tissue homeostasis, and in diverse pathological processes. Caspases and mitochondria are two key cellular components involved in the execution and regulation of apoptosis (18, 50). Caspases are a group of cysteine-proteases that are ordinarily inactive in cells as pro-enzymes but are activated upon appropriate apoptotic stimuli. Generally, the initiator caspases (e.g. 2, 8, 9, and 10) are activated when complexed with adaptor molecules, resulting in either autoprocessing due to induced proximity or holoenzyme formation (9, 18, 26, 40). The downstream effector caspases (e.g. 3, 6, and 7) are activated through proteolytic cleavage by initiator caspase(s). Effector caspases then cleave various cellular components, leading to the morphologic and biochemical phenotypes characteristic of apoptosis (11, 18).
Mitochondria also play an important role in apoptosis, as various apoptotic stimuli converge on mitochondria and lead to mitochondrial membrane permeabilization (MMP) (25, 38, 50). Upon MMP, mitochondria release a number of factors that are involved in apoptosis initiation and/or execution, such as cytochrome-c, Smac/Diablo, and AIF (Apoptosis Inducing Factor) (8, 18, 38, 50). The released cytochrome-c interacts with the adaptor protein Apaf-1 and pro-caspase-9 to form an activated complex referred to as an apoptosome, which then cleaves and activates downstream effector caspases (e.g. caspase-3) (18, 52). In contrast, AIF released from mitochondria triggers apoptosis (e.g. by inducing chromatin condensation and large-scale DNA fragmentation) independent of effector caspases (8, 31, 46, 51). This caspase-independent apoptogenic function of AIF is evolutionarily conserved, and plays an important role both in normal development and in cell death processes whereby caspases are minimally activated or inhibited (e.g. by chemical inhibitors) (8, 38).
There are two major apoptotic pathways in mammalian cells (11, 18). The extrinsic pathway is initiated by the binding of transmembrane death receptors (e.g. Fas, TNF-R1, and TRAIL receptors) with cognate extracellular ligands. Liganded receptors recruit adaptor proteins (e.g. FADD) which interact with and trigger the activation of caspase-8. Activated caspase-8 then cleaves and activates downstream effector caspases such as caspase-3. In contrast, the intrinsic pathway is characterized by disruption of mitochondria membrane integrity when cells are exposed to various stresses (e.g. DNA damaging agents). Mitochondrial membrane permeabilization (MMP) triggers apoptosis via both caspase-dependent (e.g. the cytochrome-c/caspase-9 pathway) and caspase-independent (e.g. the AIF pathway) mechanisms. Crosstalk exists between the extrinsic and intrinsic pathways, as activated caspase-8 can cleave Bid to produce truncated Bid (tBid), which then binds to mitochondria and promotes MMP (30, 32). The subsequent release of cytochrome-c from mitochondria further facilitates the apoptotic process.
MMP is regulated by the Bcl-2 family of proteins, which act upstream of mitochondria, and contain both anti-apoptotic (e.g. Bcl-2 and Bcl-xL) and pro-apoptotic members (e.g. Bak, Bax, Bid, and Bad) (3, 7). The relative balance between the pro- and anti-apoptotic members of the Bcl-2 family is critical in controlling MMP. Interestingly, a number of recent studies have shown that caspase-2 acts upstream of mitochondria and is required for MMP during stress-induced apoptosis in certain cell types (17, 27, 42). While these studies implicate caspase-2 as an initiator caspase in certain intrinsic pathway(s) of apoptosis, the mechanistic interplay between caspase-2 and members of the Bcl-2 family in controlling MMP is not yet clear (24).
The present inventors have studied the role of nuclear receptors in breast cancer cell proliferation (2). U.S. Pat. No. 6,639,064, issued Oct. 28, 2003 discloses the cloning of a novel coregulator (designated as NRIF3) which specifically interacts with and enhances the activity of ligand-bound thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) (28, 29). However, no therapeutic role was ascribed to the protein in these publications.